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Ferroptosis Contributes to Neuronal Death and Functional Outcome After Traumatic Brain Injury*

Kenny, Elizabeth M., BS1,2,3; Fidan, Emin, MD1,2,3; Yang, Qin, MD1,2,3; Anthonymuthu, Tamil S., PhD1,2,3; New, Lee Ann, BS1,2; Meyer, Elizabeth A., BS1,2; Wang, Hong, PhD4; Kochanek, Patrick M., MD2; Dixon, C. Edward, PhD2,5; Kagan, Valerian E., PhD, DSc3,6,7; Bayir, Hülya, MD1,2,3,6,7,8

doi: 10.1097/CCM.0000000000003555
Neurologic Critical Care

Objectives: Traumatic brain injury triggers multiple cell death pathways, possibly including ferroptosis—a recently described cell death pathway that results from accumulation of 15-lipoxygenase–mediated lipid oxidation products, specifically oxidized phosphatidylethanolamine containing arachidonic or adrenic acid. This study aimed to investigate whether ferroptosis contributed to the pathogenesis of in vitro and in vivo traumatic brain injury, and whether inhibition of 15-lipoxygenase provided neuroprotection.

Design: Cell culture study and randomized controlled animal study.

Setting: University research laboratory.

Subjects: HT22 neuronal cell line and adult male C57BL/6 mice.

Interventions: HT22 cells were subjected to pharmacologic induction of ferroptosis or mechanical stretch injury with and without administration of inhibitors of ferroptosis. Mice were subjected to sham or controlled cortical impact injury. Injured mice were randomized to receive vehicle or baicalein (12/15-lipoxygenase inhibitor) at 10–15 minutes postinjury.

Measurements and Main Results: Pharmacologic inducers of ferroptosis and mechanical stretch injury resulted in cell death that was rescued by prototypical antiferroptotic agents including baicalein. Liquid chromatography tandem-mass spectrometry revealed the abundance of arachidonic/adrenic-phosphatidylethanolamine compared with other arachidonic/adrenic acid-containing phospholipids in the brain. Controlled cortical impact resulted in accumulation of oxidized phosphatidylethanolamine, increased expression of 15-lipoxygenase and acyl-CoA synthetase long-chain family member 4 (enzyme that generates substrate for the esterification of arachidonic/adrenic acid into phosphatidylethanolamine), and depletion of glutathione in the ipsilateral cortex. Postinjury administration of baicalein attenuated oxidation of arachidonic/adrenic acid-containing-phosphatidylethanolamine, decreased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells in the hippocampus, and improved spatial memory acquisition versus vehicle.

Conclusions: Biomarkers of ferroptotic death were increased after traumatic brain injury. Baicalein decreased ferroptotic phosphatidylethanolamine oxidation and improved outcome after controlled cortical impact, suggesting that 15-lipoxygenase pathway might be a valuable therapeutic target after traumatic brain injury.

1Department of Critical Care Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA.

2Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA.

3Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA.

4Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA.

5Department of Neurosurgery, School of Medicine, University of Pittsburgh, Pittsburgh, PA.

6Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA.

7Laboratory of Navigational Redox Lipidomics, Institute of Regenerative Medicine, IM Sechenov Moscow State Medical University, Moscow, Russia.

8Children’s Neuroscience Institute, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA.

*See also p. 480.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ccmjournal).

Supported, in part, by National Institutes of Health grants (NS061817, NS076511, AI068021, and NS079061).

Drs. Yang, Mayer, Kochanek, Dixon, Kagan, and Bayir received support for article research from the National Institutes of Health (NIH). Dr. Kochanek’s institution received funding from the NIH; he received funding from Society of Critical Care Medicine (Editor-in-Chief of Pediatric Critical Care Medicine) and from serving as an expert witness and a visiting professor/grand rounds speaker (travel/compensation); and he disclosed other funding separate from that reported in this study by both the NIH, the U.S. Department of Defense, and the state of Pennsylvania. Dr. Kagan disclosed government work. The remaining authors have disclosed that they do not have any potential conflicts of interest.

For information regarding this article, E-mail: bayihx@ccm.upmc.edu

Traumatic brain injury (TBI) is a critical health concern that results in significant morbidity, mortality, and financial cost worldwide (1–3). In the United States alone, TBI accounts for approximately 2.5 million emergency department visits, 280,000 hospitalizations, and 52,000 deaths annually (1). Direct and indirect costs of TBI in the United States approach $76.5 billion per year, with severe TBI accounting for 90% of this medical spending (4 , 5). Despite the high burden of severe TBI, effective neuroprotective therapies are lacking with no clear improvement in overall outcome in more than two decades (6).

Neuronal death contributes to neurologic deficit after TBI, and thus it is a reasonable therapeutic target. Ferroptosis is a recently described, regulated cell death pathway that results from accumulation of lipid oxidation products (Fig. S1, Supplemental Digital Content 1, http://links.lww.com/CCM/E186legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197) (7 , 8). We recently showed that 15-lipoxygenase-mediated oxidation of phosphatidylethanolamines, specifically those containing arachidonic (AA) and adrenic acid (AdA), is a key step in ferroptosis execution (9, 10). Complex formation between 15-lipoxygenase and phosphatidylethanolamine-binding protein 1 (PEBP1) results in a change in 15-lipoxygenase substrate specificity from free to esterified AA (10). Acyl-CoA synthetase long-chain family member 4 (ACSL4) contributes to ferroptosis by generating substrate for the esterification of AA/AdA into phosphatidylethanolamine (11). Glutathione peroxidase 4 (GPX4) is the only member of the GPX family that can reduce hydroperoxy-phospholipids to nontoxic hydroxy-phospholipids utilizing glutathione as the reducing equivalent (12). Insufficiency of GPX4 or glutathione results in ferroptosis due to the accumulation of oxidized phosphatidylethanolamine (PEox).

Dysregulation of key components of ferroptotic machinery including glutathione depletion and lipid oxidation has been observed after experimental (13) and clinical (14) TBI. We therefore reasoned that phosphatidylethanolamine peroxidation and ferroptotic death may be an important pathogenic pathway in TBI. We first characterized the neuroprotective effects of ferroptosis inhibition following pharmacologic induction of ferroptosis and in vitro TBI. Using a liquid chromatography tandem-mass spectrometry (LC-MS/MS)-based redox lipidomics approach, we investigated ferroptotic PEox death signals in the pericontusional cortex after controlled cortical impact (CCI) in mice. We further examined the role of ferroptosis through quantification of key ferroptotic enzymes and glutathione in the ipsilateral cortex. Finally, we tested the ability of baicalein, a 12/15-lipoxygenase inhibitor, to attenuate ferroptotic signaling after CCI.

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MATERIALS AND METHODS

Materials

A GPX4-specific inhibitor, RAS-selective lethal compound 3 (RSL3), was purchased from Selleck Chemicals (Houston, TX). Unless otherwise indicated, reagents were purchased from Sigma-Aldrich (Saint Louis, MO).

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In Vitro TBI Model

HT22 cells were a generous gift from Dr. David Schubert (The Salk Institute, La Jolla, CA). These cells were previously shown to undergo ferroptosis following RSL3 treatment (10). Cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 1% penicillin-streptomycin (37°C, 5% CO2). In vitro TBI was performed using a well-established model of mechanical stretch injury (15). At 24 hours before stretch, cells were trypsinized and seeded (400,000 cells/well) on silicone membranes within custom-made stainless-steel wells. Wells were fitted into the stretch apparatus, and a severe stretch injury (strain rate, 10/s; membrane deformation, 50%; peak pressure, 3–4 psi) was applied to simulate a similar strain field to that of our in vivo TBI model. Data from at least three independent experiments are presented in Figure 1 and Figure S3 (Supplemental Digital Content 4, http://links.lww.com/CCM/E189legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197).

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Cell Death Assessment

Cell death was assessed by measuring activity of lactate dehydrogenase released into the cell culture media using the Cytotoxicity Detection Kit as per manufacturer’s instructions (Promega, Madison, WI).

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In Vivo TBI Model

Experiments were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Care and handling of the animals were in accord with National Institutes of Health Guidelines. Adult male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were subjected to severe TBI as previously described (16). Anesthesia was induced with 3% isoflurane in nitrous oxide:oxygen (70:30) and maintained with 1.5% isoflurane via nose cone, and rectal temperature was maintained at 37°C ± 0.5°C. Animals were placed onto a stereotaxic frame and secured with ear bars. The bone overlying the left parietal cortex was removed. CCI was produced using a flat 3-mm pneumatically driven impactor tip (6.0 ± 0.2 m/s, 50 ms dwell time, 1.4 mm depth). The bone flap was replaced and sealed, and the scalp incision was closed. Mice were monitored with supplemental oxygen (100%) for 1 hour before returning to their cages. Baicalein (50 mg/kg) was administered via intraperitoneal injection (17–19) at 10–15 minutes post-CCI. Baicalein was dissolved in cremophor El and ethanol (CEE) solution and diluted with normal saline (NS) (CEE:NS, 1:3, v/v). Separate experiments were performed for biochemical, histologic, and functional outcome testing with sample sizes of 4–6 per group for the first two outcomes and 9–13 for the functional outcome. The number of animals used in each experiment is shown in figure legends (Figs. 2–4; and Fig. S2, Supplemental Digital Content 3, http://links.lww.com/CCM/E188; Fig. S3, Supplemental Digital Content 4, http://links.lww.com/CCM/E189; Fig. S4, Supplemental Digital Content 5, http://links.lww.com/CCM/E190; Fig. S6, Supplemental Digital Content 7, http://links.lww.com/CCM/E192; Fig. S8, Supplemental Digital Content 9, http://links.lww.com/CCM/E194; and Fig. S9, Supplemental Digital Content 11, http://links.lww.com/CCM/E196—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197).

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Lipid Extraction and LC-MS/MS Analysis

Lipid extraction of the pericontusional cortex was achieved using the Folch method. Total phosphate content of the lipid extracts was quantified using a micromethod (20). Extracted lipids (≈30 nmol of total phospholipids) were added to a glass tube with 7.5 pmol of each deuterated internal standard (Table S1, Supplemental Digital Content 2, http://links.lww.com/CCM/E187) and dried under nitrogen flow. The dried film was dissolved in 15 μL of solvent A (hexane:2-propanol:water, 430:570:10, v/v/v), and 5 μL of the reconstituted sample was injected in duplicate into the LC-MS/MS system for phospholipid analysis. LC-MS/MS analysis was performed using a Dionex UltiMate 3000 RSLCnano System coupled online to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA) using a normal-phase Silica (2) column (Luna 3 µm, 100 Å, 150 × 2.1 mm) (Phenomenex, Torrance, CA) as previously described (10). The MS data were analyzed using Compound Discoverer software (Thermo Fischer Scientific, San Jose, CA) as previously described (10).

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Protein and Glutathione Measurements

Ipsilateral cortex was sonicated in lysis buffer (Tris base 30 mM, sodium chloride 150 mM, Triton-X 0.5%, pH 7.4) with protease and phosphatase inhibitors (Thermo Scientific-PIERCE, Rockford, IL). Samples were centrifuged (10 min, 16,000g, 4°C), and supernatants were collected for protein and glutathione measurements. Western blotting was performed using the following antibodies: GPX4 (1:500, ab125066), 15-lipoxygenase 2 (1:200–500, sc-67143), ACSL4 (1:500, sc-13450), actin (1:1000, A3854), and horseradish peroxidase–conjugated antibodies (1:2000, ab97023/ab97051/ab6721). Bands were detected using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA) and the ChemiDoc XRS+ System (Bio-Rad, Hercules, CA). NIH ImageJ software (National Institute of Health, Bethesda, MD) was used for densitometry analyses. Glutathione was measured using a fluorescent assay with ThioGlo-1 (Covalent Associates, Woburn, MA) as previously described (13).

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Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling Staining

Mice were perfused with heparinized saline followed by 4% paraformaldehyde. Brains were extracted, postfixed (24 hr, 4% paraformaldehyde), and embedded in paraffin. Using a Leitz 1512 Microtome (Leica Biosystems, Buffalo Grove, IL), multiple 5-μm coronal sections of the dorsal hippocampus (200-μm intervals) were collected. The ApopTag Peroxidase In Situ Apoptosis Detection Kit (S7110) was used to detect terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positivity according to the manufacturer’s instruction (EMD Millipore, Billerica, MA). Briefly, tissue sections were deparaffinized and incubated with terminal deoxynucleotidyl transferase enzyme and a mixture of digoxigenin-labeled and unlabeled nucleotides. Samples were subsequently incubated with fluorescein-conjugated antidigoxigenin antibody and counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Two consecutive coronal sections of the dorsal hippocampus underlying the cortical contusion were stained, imaged, quantified, and averaged per animal. TUNEL-stained neurons were counted in the entire anatomic Cornu Ammonis (CA) 1, CA2, CA3, and dentate gyrus regions and normalized to area. TUNEL positivity was quantified per square millimeter in CA1, CA2, CA3, and dentate gyrus sections stained with TUNEL and DAPI (EMD Millipore, Billerica, MA). Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Melville, NY), and analysis was performed using NIS Elements software (Nikon, Melville, NY).

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Morris Water Maze Performance

The Morris water maze (MWM) task was used to assess acquisition of spatial learning (21). The maze consisted of a plastic pool (83 cm diameter, 60 cm height) filled with water (26°C ± 1°C) to a depth of 29 cm and placed in a room with salient visual cues that remained unchanged. A clear Plexiglass platform (10 cm diameter, 28 cm height) was positioned 15 cm from the wall of the pool in the southwest quadrant for all trials. Acquisition of spatial learning was evaluated through four daily trials on postoperative day (POD) 10–14 based on a lack of motor deficits in open field test between sham and CCI groups by POD 5 (Fig. S2, Supplemental Digital Content 3, http://links.lww.com/CCM/E188—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197). Trials lasted until the mouse reached the hidden platform or until 120 seconds had elapsed. Mice that failed to find the platform were manually guided to it, and all animals were kept on the platform for 10 seconds after each trial. Mice were placed in a heated incubator for 5 minutes between trials. On POD 15, the platform was made visible (2 cm above the water), and the task was repeated to assess deficits in visual and motor function. The ANY-Maze video tracking system (San Diego Instruments, San Diego, CA) was used to record MWM parameters.

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Statistical Analysis

Statistical analyses of biochemical and histologic data were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA). Data were analyzed using Student t test or analysis of variance (ANOVA) with Tukey post hoc analysis for two- or multigroup comparisons, respectively. Time to hidden platform endpoint in MWM task was analyzed with SAS 9.4 (SAS Institute, Cary, NC) software. Data were summarized as mean ± SD for each group (i.e., sham, vehicle-CCI, or baicalein-CCI) on each day. PROC TRANSREG in SAS 9.4 (SAS Institute, Cary, NC) software was used to check if a Box-Cox transformation (22) was necessary to make the data normally distributed. It was found that the data could be regarded as normally distributed after log transformation. To test whether the three groups had the same rate of decrease over the 5 days, a linear mixed model was built on the log-transformed data, where group and day and their interaction were used as fixed effects, and animal ID was used as a random effect. For time to platform under visible condition, data were summarized as mean ± SD for each group. The comparison between groups was performed by one-way ANOVA followed by Tukey multiple comparisons on log-transformed data. Statistical significance was set at p value less than 0.05, and data are expressed as mean ± SD.

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RESULTS

Ferroptosis Inhibition Rescues HT22 Neurons From In Vitro TBI

We first evaluated whether antiferroptotic agents could rescue neurons from death induced by proferroptotic conditions (glutathione or GPX4 insufficiency) or in vitro TBI. HT22 cells were exposed to RSL3 (inhibitor of GPX4) resulting in increased cell death that was rescued by inhibitors of key ferroptotic regulatory steps: ferrostatin-1 (lipid radical-trapping antioxidant), triacsin C (ACSL4 inhibitor), and baicalein (12/15-lipoxygenase inhibitor) (Fig. 1A; and Fig. S1, Supplemental Digital Content 1, http://links.lww.com/CCM/E186—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197). Similar results were obtained with glutamate or erastin (inhibitors of cystine/glutamate antiporter which result in depletion of intracellular cysteine—the rate-limiting substrate of glutathione synthesis) and L-buthionine sulfoximine (inhibitor of rate-limiting enzyme in glutathione synthesis) (Fig. S1, Supplemental Digital Content 1, http://links.lww.com/CCM/E186; and Fig. S3, A–C, Supplemental Digital Content 4, http://links.lww.com/CCM/E189—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197). Similarly, in vitro TBI elicited by mechanical stretch resulted in increased cell death that was significantly reduced with administration of ferrostatin-1, triacsin C, baicalein, or liproxstatin-1 (lipid radical-trapping antioxidant) (Fig. 1B).

Figure 1

Figure 1

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Phosphatidylethanolamine in Mouse Cortex Contains More Arachidonic/AdA Than Other Phospholipid Classes

We previously showed that oxidation of AA/AdA-phosphatidylethanolamine is a key step in ferroptosis execution (9). Therefore, we next examined the cortical phospholipidome of uninjured mice using high-resolution LC-MS/MS. Among the identified phospholipid classes (Fig. S4A, Supplemental Digital Content 5, http://links.lww.com/CCM/E190—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197), abundance of AA/AdA-phosphatidylethanolamine was higher than all other classes of AA/AdA-containing phospholipids (Fig. S4B, Supplemental Digital Content 5, http://links.lww.com/CCM/E190—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197), demonstrating an abundance of precursor for ferroptosis execution in the brain. The identity of AA/AdA-phosphatidylethanolamine species was confirmed through fragmentation analysis (Fig. S5, Supplemental Digital Content 6, http://links.lww.com/CCM/E191—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197).

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CCI Results in Phospholipid, Protein, and Glutathione Changes Consistent With Ferroptosis Activation

As we had observed an abundance of AA/AdA-phosphatidylethanolamine in mouse cortex, we next examined phosphatidylethanolamine oxidation in naive and CCI-injured animals at 4 hours after impact. LC-MS/MS analysis showed a significant increase in total phosphatidylethanolamine oxidation (Fig. 2A) and individual oxidized phosphatidylethanolamine species (Fig. 2B) in pericontusional versus naive cortex. Identities of the significantly elevated PEox species (Fig. S6, Supplemental Digital Content 7, http://links.lww.com/CCM/E192—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197), confirmed with fragmentation analyses (Fig. S7, Supplemental Digital Content 8, http://links.lww.com/CCM/E193—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197), were consistent with ferroptotic mediators previously identified by our group (9). Fragmentation analyses of PEox demonstrated a predominance of 15-lipoxygenase products including 15-hydroperoxyeicosatetraenoic acid.

Figure 2

Figure 2

In line with the predominance of 15-lipoxygenase-generated oxidized lipid products, 15-lipoxygenase 2 expression was significantly elevated post-CCI versus naive (Fig. 3, A and B). ACSL4 was also significantly elevated after injury (Fig. 3, A and B). Expression of GPX4 remained unchanged (Fig. 3, A and B), and glutathione levels were significantly decreased after injury (Fig. 3C). Overall, the protein and glutathione profile in injured brain reflected a shift toward a pro-oxidative lipid environment (15-lipoxygenase 2 and ACSL4) without a compensatory increase in lipid-hydroperoxide reducing capacity (GPX4 and glutathione).

Figure 3

Figure 3

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Lipoxygenase Inhibition Results in Histologic Neuroprotection and Decreased Phosphatidylethanolamine Oxidation After CCI

With strong evidence for a role of 15-lipoxygenase in the pathogenesis of CCI, we sought to determine whether lipoxygenase inhibition via baicalein affected neuronal death and ferroptotic phosphatidylethanolamine oxidation. Baicalein-treated animals demonstrated significantly less hippocampal TUNEL positivity than vehicle-treated mice post-CCI (Fig. 4 A–C). Because both ferroptotic (12) and apoptotic cells can be detected with TUNEL, we assessed cardiolipin oxidation. Oxidation of cardiolipin through cytochrome C/cardiolipin complex by hydrogen peroxide is known to result in the release of proapoptotic signals (23). Nonoxidized cardiolipin decreased, and oxidized cardiolipin increased in the cortex after CCI. However, these changes in cardiolipin oxidation were not affected by baicalein administration (Fig. S8, Supplemental Digital Content 9, http://links.lww.com/CCM/E194—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197). Conversely, baicalein significantly attenuated phosphatidylethanolamine oxidation, particularly proferroptotic trioxygenated phosphatidylethanolamine (38:5) and trioxygenated phosphatidylethanolamine (40:5) species (9 , 10) after CCI versus vehicle (Fig. 2, A and B; and Fig. S6, Supplemental Digital Content 7, http://links.lww.com/CCM/E192—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197).

Figure 4

Figure 4

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Lipoxygenase Inhibition Results in Functional Improvement After CCI

We next evaluated the effect of baicalein on functional outcome after CCI using the MWM task. Based on the linear mixed model on log (time to platform), the interaction of group and day was not significant (p = 0.5797); thus, we removed the interaction term from the model and refit the data. We found that the time to platform in each group correlated with the day and with the following relationship (where numbers in the parentheses are the corresponding SEs):

Sham: log (time to platform) = 3.72 (0.13) – 0.22 (0.03) × day

Vehicle-CCI: log (time to platform) = 4.31 (0.12) – 0.22 (0.03) × day

Baicalein-CCI: log (time to platform) = 3.91 (0.12) – 0.22 (0.03) × day

To test for the average difference between the three groups, we ran F tests in this model and obtained the results summarized in Table 1. Overall, the three groups did not have significant difference in slope of change, and over the 5 days, the vehicle-CCI group had significantly longer time to platform than the sham and the baicalein-CCI groups, but there was no difference between sham and baicalein-CCI (Table 1 and Fig. 4D). Under visible platform conditions, the three groups did not have significant difference (Table S2, Supplemental Digital Content 10, http://links.lww.com/CCM/E195) indicating a lack of motor or visual deficit. A one-way ANOVA for time spent in the target quadrant during probe trial revealed a significant group main effect (p = 0.0009). Post hoc analysis showed that CCI-injured groups spent significantly less time in the target quadrant than shams, but the baicalein-CCI group was not different than the vehicle-CCI group (Fig. S9, Supplemental Digital Content 11, http://links.lww.com/CCM/E196—legend, Supplemental Digital Content 12, http://links.lww.com/CCM/E197).

TABLE 1

TABLE 1

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DISCUSSION

Since the recent identification of ferroptosis as a distinct cell death pathway, its role in disease pathogenesis is increasingly recognized (10 , 24–27). Several lines of evidence suggest that ferroptosis may be particularly important in the brain: 1) brain contains the highest polyunsaturated fatty acid content in the body, and thus possesses abundant precursor for lipid peroxidation (28). 2) Glutathione depletion and lipid peroxidation are associated with neurodegenerative disease including TBI (29). 3) Genetic deficiency of GPX4 results in neuronal death in vitro and in vivo (30–33). Here, we provide evidence that ferroptosis contributes to neuronal death and functional outcome after TBI. To our knowledge, this study is the first study directly examining ferroptosis inhibition in both in vitro and in vivo TBI.

Several studies have evaluated ferroptosis in models of acute brain injury. The earliest study used a rat organotypic hippocampal slice culture model to examine ferroptosis after glutamate excitotoxicity (7)—a key phenomenon in neurodegeneration and brain trauma (34). Coincubation with ferrostatin-1 or ciclopirox (iron chelator) significantly attenuated glutamate-induced cell death (7). Neuroprotective effects of ferroptosis inhibition were recently shown in intracerebral hemorrhage models (35 , 36). Interestingly, lipid oxidation was reported to have a detrimental effect on the transition of astrocytes and fibroblasts into neurons after TBI (37). Treatment with liproxstatin-1 enhanced in vitro astrocyte conversion efficiency, whereas α-tocotrienol enhanced conversion efficiency in a penetrating brain injury model. Finally, we recently reported that 15-lipoxygenase expression and activity were increased and GPX4 expression and activity were decreased with colocalization of 15-lipoxygenase and PEBP1 in the hippocampus after CCI in a developmental TBI model in postnatal day 17 rats (10). Furthermore, we showed that ferroptotic PEox species were increased in the cortex (10). Together these data indicated that biomarkers of ferroptosisincrease after CCI in the immature brain. Additionally, this study established that formation of the complex of 15-lipoxygenase and PEBP1 changed the substrate specificity of 15-lipoxygenase from free-AA to AA-esterified into phosphatidylethanolamine. The current study extends these observations to adult mice and shows that baicalein posttreatment decreased ferroptotic PEox after CCI. In addition, we show that ferroptosis inhibitors, including baicalein, attenuate mechanical stretch-induced death in HT22 hippocampal neuronal cells suggesting that this in vitro TBI model might be useful for therapeutic screening of drugs targeting ferroptosis. Furthermore, future studies evaluating the quantitative contribution of this pathway compared with other apoptotic and necrotic death pathways could be revealing (15 , 38 , 39).

Although several studies have shown neuroprotective effects of ferrostatin-1 in vivo, ferrostatin-1 has limited biological availability (40), and it is possible that targeting upstream in this pathway at the level of lipid peroxidation may be more effective. Although cyclooxygenases and cytochrome P450 can catalyze lipid oxidation (41), to date only a role of the lipoxygenase family has been demonstrated in ferroptosis (31 , 42 , 43). In humans, the lipoxygenase family comprised of six functional isoforms which differ in their preferred carbon for oxidation with respect to AA (44). Species and tissue variation of lipoxygenase isoforms (44) and lack of structural information for human lipoxygenase isoforms (26) make pharmacologic inhibition of lipoxygenase as an antiferroptotic strategy challenging. Furthermore, specific disruptors of 15-lipoxygenase-PEBP1 complexes might be more effective as antiferroptotic compounds. Isoform-specific lipoxygenase inhibitors are under development (45) and may inhibit ferroptosis more efficiently than nonspecific or pan-lipoxygenase inhibitors.

As 15-lipoxygenase 2-specific inhibitors are currently unavailable, we assessed the effects of baicalein in our TBI model. Baicalein is a polyphenolic flavonoid and 12/15-lipoxygenase inhibitor (46). Compared with synthetic, antiferroptotic agents such as liproxstatin-1 and ferrostatin-1, baicalein is an herbal supplement found in natural products, which may more rapidly facilitate its use in clinical settings. The importance of 12/15-lipoxygenase and the protective effects of baicalein are well studied in models of focal cerebral ischemia (47 , 48). Although one study has demonstrated improved motor function and decreased number of FluoroJade B-positive neurons with baicalein treatment immediately post-CCI in rats, this study focused on an anti-inflammatory mechanism to explain its protective effects (19).

To our knowledge, our study represents the first analysis of baicalein administration for ferroptosis inhibition after TBI. The versatile chemical structure of baicalein makes it a non-narrowly specific lipoxygenase inhibitor with several alternative mechanisms of action (19 , 49). However, given that 15-lipoxygenase is responsible for the production of proferroptotic PEox species (9–11) and our redox lipidomics data demonstrate that baicalein attenuated accumulation of these PEox after CCI, without affecting proapoptotic cardiolipin oxidation, we suggest that baicalein’s effects are due, at least in part, to its inhibitory effects on 15-lipoxygenase. Currently hydroperoxy-arachidonoyl- and adrenoyl-phosphatidylethanolamine species are the most reliable quantitative predictive markers of ferroptosis (8). Although we observed functional and histologic protection with early (10–15 min) baicalein administration post-CCI, delayed drug delivery may be more practical in clinical settings.

Although we observed effects of baicalein on proferroptotic PEox generation, hippocampal cell death, and spatial memory acquisition in MWM task, baicalein did not affect memory retention assessed by the probe trial or lesion volume in our study. This could be due to multiple mechanisms of action of baicalein as well as suboptimal drug dosing or timing (19 , 49). Bilateral hippocampal lesions are required to produce significant MWM deficits in rodents in the absence of trauma (50). However, numerous studies have shown that unilateral CCI leads to neuronal death in the hippocampus (primarily in the CA3 region) and results in MWM deficits (51 , 52). Furthermore, previous studies have shown improvement in MWM performance without an effect on lesion volume after CCI with treatment (53 , 54). This is in line with the data showing that MWM has important nonhippocampal components (55). Indeed even the mice that have bilateral intrahippocampal excitotoxic lesions can significantly improve in acquisition of spatial memory during training (55). In recent years, the role of entorhinal cortex in spatial learning and memory has been elucidated. Medial entorhinal cortex, especially in layer 2, has been shown to have place cells and communicating with place cells in the hippocampus (56 , 57). Entorhinal cortex is one of the affected regions after CCI in mice (52). Our redox lipidomics analysis of the pericontusional cortex showed that baicalein attenuated CCI-induced proferroptotic PEox species. In addition to hippocampus and entorhinal cortex, white matter injury can also affect MWM outcome after CCI in mice (58). White matter lesions may affect procedural learning component of MWM (59 , 60).

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CONCLUSIONS

In conclusion, ferroptosis inhibition may represent a viable therapeutic target for neuroprotection following TBI. Here we demonstrated changes in phosphatidylethanolamine oxidation, protein expression, and glutathione levels that were consistent with ferroptosis activation post-CCI. Baicalein administration significantly reduced cell death following ferroptosis induction, in vitro TBI, and in vivo TBI. Furthermore, baicalein attenuated phosphatidylethanolamine oxidation and provided histologic and cognitive protection postinjury.

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ACKNOWLEDGMENT

We thank Henry Alexander for technical assistance with controlled cortical impact experiments.

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Keywords:

baicalein; cell death; ferroptosis; lipid oxidation; lipoxygenase; traumatic brain injury

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